The discharge of Ca2+ from intracellular stores is crucial to the regulation of fertilization, secretion, muscle contraction, neuronal function, immune responses, programmed cell death and many other diverse cellular processes. Binding of the intracellular messenger D-myo-inositol 1,4,5-trisphosphate (IP3) by a specific receptor/Ca2+ ion channel (IP3R) mediates the mobilization of Ca2+ from internal stores. The multifunctional serine/threonine protein kinase Akt acts downstream of the lipid kinase phosphoinositide 3-kinase and functions as an essential mediator in many cellular responses including cell-cycle regulation, modulation of intermediary metabolism, cell survival and transcriptional regulation. Thus IP3Rs and Akt kinase are at key nodes in intracellular signaling networks. This grant has as its starting point that all three isoforms of IP3Rs have a consensus sequence site for Akt phosphorylation in the C-terminal tail and our preliminary data indicate that this site can be phosphorylated by Akt kinase in vitro and in vivo. The objective of this proposal is to study the mechanism of the interaction between Akt kinase and IP3Rs and to assess the biological role of this phosphorylation. The specific aims of the proposed study are: I. To study the mechanism of Akt phosphorylation of IP3R channels The specificity with respect to IP3R and Akt kinase isoforms will be examined. The role of PP1, PP2A and other phosphatases in IP3R phosphorylation will be investigated. Immunofluorescence and subcellular fractionation will be used to test the hypothesis that a substantial amount of Akt kinase activation occurs at the ER or that Akt translocates to the ER membrane. II. To determine the biological consequences of IP3R phosphorylation at the Akt site. COS cells and DT-40 ZP3R knock-out cells will be used to examine the functional consequences of Akt phosphorylation by monitoring Ca2+ fluxes. The hypothesis that cytochrome c binding to the IP3R is inhibited by Akt phosphorylation will be tested. Stable DT-40 cell lines expressing IP3R mutants of the Akt phosphorylation site will be used to determine if Akt phosphorylation influences the ability of the cells to undergo Ca2+ -dependent gene activation or apoptosis in response to IgM. This pilot and feasibility proposal is intended to assess the physiological significance of Akt kinase phosphorylation of IP3Rs. Understanding the 'crosstalk' between Ca2+ signaling and the Akt pathways may yield hitherto unrecognized insights into both pathways. The studies proposed in this application are significant since both signaling systems play important roles in many clinically relevant processes such as insulin and growth factor signaling, angiogenesis and oncogenesis.